A treatment (cleanup) for restoring a natural environment polluted with a harmful substance to its unpolluted state, which includes no harmful substance, using a microorganism (such as a soil-improving bacterium) is called bioremediation. In the bioremediation, in order to grasp the degree of progress of the cleanup, it is essential to accurately monitor a microorganism spread in an environment. Further, it is necessary to accurately grasp the number of residual microorganism cells at a time when the cleanup is completed.
An existing procedure includes preparing total DNA in an environment (environmental DNA) where a microorganism is spread and performing a quantitative polymerase chain reaction (PCR) using, as a primer, a DNA sequence specific to the microorganism to roughly grasp the number of the microorganism cells (for example, see Non-patent Document 1). However, it is difficult to accurately grasp the number of the microorganism cells because the primer to be used in the quantitative PCR may react with non-specific DNA sequence (DNA sequences other than DNA sequences of interest in the environment, for example).
Hitherto, many reports have been made on technologies for improving primer specificity in PCR and programs for the technologies (for example, see Non-patent Documents 2 and 3). However, all the technologies are specialized for an organism which has been isolated and cultured. If the organism which has been isolated and cultured is used, probability of a reaction of the primer designed with a region other than a region to be treated can be estimated by a search of the genome. Meanwhile, in the environmental DNA in which a large indefinite number of organisms coexist, it is very difficult to estimate the specificity of the primer.
Further, the law required by the international treaty relating to a rule of handling of recombinant organisms (Cartagena Protocol) (hereinafter, sometimes referred to as “Cartagena Law”) prohibits spread of recombinant microorganisms to open environments. Therefore, it is impossible to make an appropriate artificial DNA sequence which shows high primer specificity and introduce the sequence into a microorganism to be used in bioremediation or the like without careful consideration. Meanwhile, there has not been proposed a technology for introducing an artificial primer sequence into a microorganism to be used in bioremediation in such a range that the microorganism does not fall within the category “recombinant organism” specified by the Cartagena Law.
The bioremediation has advantages in that the cost and energy consumption are low as compared to a physicochemical cleanup method and that the method does not burden an ecosystem because the technology is mild, for example. On the other hand, sufficient findings on effects of spread of a microorganism for cleanup on an environment have not been obtained. Therefore, to grasp not only the degree of progress of cleanup but also the degree of dispersion of the microorganism spread and effects on the ecosystem in the field of spread as well, means for accurately monitoring has been strongly desired.